Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. Coxiellas former obligate intracellular nature significantly impeded genetic characterization of putative virulence factors. However, the seminal advance of host cell-free (axenic) growth of Coxiella in acidified citrate cysteine medium (ACCM) enabled us to quickly develop shuttle vector, transposon mutagenesis, inducible gene expression, and targeted gene deletion technologies for pathogen genetic manipulation. We have also recently developed a Luciferase gene reporter system that can by used to monitor developmentally-regulated gene expression and pathogen stress responses. The repertoire of Coxiella genetic tools now allows traditional mutation and complementation strategies for virulence factor discovery. Indeed, we have constructed over 30 knockout strains including those with deletions in genes encoding components of the Dot/Icm type IVB secretion system (T4BSS) and secreted proteins. These studies have confirmed that T4BSS function is critical for Coxiella growth in macrophages. Mutational analysis has also identified several T4BSS effector proteins that are required for optimal growth of mammalian cells. The two-component regulatory system PmrA-PmrB is predicted to regulate type IVB secretion in Coxiella. The response regulator PmrA binds a regulatory element upstream of target genes to affect gene transcription. To probe PmrAB-mediated gene regulation in Coxiella, we used our newly developed luciferase-based gene reporter system and a pmrA deletion mutant to show that PmrA is a critical positive regulator of the Dot/Icm T4BSS and several secreted effectors. Consequently, Coxiella &#916;pmrA has a severe growth defect in mammalian cells with little to no replication in LAMP3-positive PVs. Mutation of predicted regulatory elements defined nucleotides required for promoter recognition by PmrA. Additionally, RNA sequencing and mass spectrometry revealed new Dot/Icm substrates and several PmrA-regulated genes potentially involved in virulence that lack a PmrA regulatory element. Thus, the PmrAB TCS may crosstalk with other TCSs and/or indirectly control expression of additional regulatory systems. This study begins to unravel the transcriptional regulatory networks associated with Coxiella virulence. Central to pathogenesis by Coxiella is the ability to proliferate in a phagolysosome-like PV of human macrophages. The specific adaptations that enable growth in this normally toxic environment are largely unknown. Therefore, to gain insight into intracellular growth strategies of Coxiella, we used microarrays to compare the transcriptomes of organisms propagated in primary human monocyte derived macrophages (HMDM) and ACCM. Messenger RNA was isolated from Coxiella at 4 days post-infection of macrophages and inoculation of ACCM, a time point that coincides with late log phase in both culture conditions. Upregulated genes during growth in HMDM include those encoding oxidative stress response proteins and nutrient transporters. Collectively, these data suggest the Coxiella PV is a nutritionally limited and oxidative environment relative to ACCM. This report provides new information on specific transcriptional responses by Coxiella that enable successful parasitism of macrophages. The only genetic lesions known to result in attenuated Coxiella virulence in an animal model of human Q fever are associated with defective lipopolysaccharide (LPS) synthesis. Virulent phase I organisms with full-length LPS transition to avirulent phase II organisms with severely truncated LPS upon repeated in vitro passage. Given the critical role of LPS in Coxiella virulence, it is important to understand the molecular basis of phase variation. The high passage phase II isolates in our stock collection are not clonal and contain a small subpopulation of Coxiella still expressing full-length phase I LPS. The resulting mixed genotype complicates identification of indels (insertions/deletions) strictly associated with phase variation. To circumvent this problem, we used micromanipulation to isolate clonal phase II populations of high passage Nine Mile, Australia and California isolates. By hybridizing their genomic DNA to a high-density microarray that contains probe sets encompassing the entire genome of the Nine Mile phase I isolate, common indels in phase II organisms were identified that may account for defective LPS biosynthesis. Coxiella undergoes an intracellular biphasic developmental cycle that generates two distinct morphological variants that can be distinguished by ultrastructure and protein composition. Small cell variants (SCV) do not replicate, contain condensed chromatin, and are considered extracellular survival forms. SCV differentiate into replicative large cell variants (LCV) with dispersed chromatin. Transition of LCV back to SCV occurs coincident with entry of Coxiella into stationary growth phase with nearly homogeneous SCV present upon extended incubation (2 to 4 weeks) of infected cell cultures. As an amenable model to help us better understand the biological relevance of Coxiella differentiation, we established that SCV/LCV transitions are recapitulated by organisms growing in the second-generation axenic media, ACCM-2. This discovery enables studies of Coxiella developmental biology without experimental difficulties encountered with host cell-propagated bacteria. Comparative transcriptomics and proteomics of LCV and SCV have now revealed molecular determinants of morphological differentiation that likely contribute to the unique biological characteristics of cell forms.